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HEALTH CONSULTATION
CRYSTAL LAKE BIKE TRAIL
BEULAH, BENZIE COUNTY, MICHIGAN

SUMMARY

A former railroad bed that is being converted to a recreational trail crosses residential yards. The soil in the rail bed was found to contain levels of arsenic and benzo(a)pyrene above state screening levels. One resident experienced symptoms suggestive of arsenic toxicity. Her personal physician diagnosed her with arsenic toxicosis based on the symptoms and on laboratory results indicating high arsenic content in her hair. Another resident has leukemia and asks if the diagnosis is attributable to the benzo(a)pyrene.

Based on soil sampling data, the arsenic and benzo(a)pyrene in the railroad bed pose no apparent public health hazard. While the first resident's symptoms and hair testing results were suggestive of excessive arsenic exposure, adverse health effects would not be expected to occur, based on soil data. Nonetheless, because much of the soil in the rail bed exceeds the state's screening level for arsenic, the state regulatory agency should consider remediating the site.

PURPOSE AND HEALTH ISSUES

The purpose of this health consultation is to address the health issues concerning the Crystal Lake Bike Trail. The document will discuss the findings from environmental sampling as well as from biomarker (hair) analysis. Based on the findings, the Michigan Department of Community Health (MDCH) will form a health conclusion and make recommendations. The document will not discuss in detail the political background of ownership of and responsibility for the railroad right-of-way/trail.

BACKGROUND

In November 2001, the Michigan Department of Environmental Quality (MDEQ) requested a health consultation from MDCH regarding specific health-related questions from residents whose yards contain portions of a former railroad bed that is being converted into a recreational trail. The area of interest is located along the southeast shore of Crystal Lake in Beulah, Benzie County, Michigan (Figure 1). The MDEQ tested the railroad bed soil, in response to a request from a local citizen. Analytical results revealed concentrations of arsenic and benzo(a)pyrene in excess of state screening levels (see Discussion section).

Beginning in 1997, a female resident experienced hypopigmentation (lack of pigmentation of the skin) and dermatitis (a rash) with oozing sores on her arms. She has since become photosensitive and cannot be exposed to sunlight without the exposure resulting in a skin reaction. She and her husband have owned their property by Crystal Lake for 20 years and constructed their home there. During construction, they did not have access to running water and consequently could not wash as frequently as they otherwise would have. They used water from their well, taken from a spigot at the wellhead, for drinking. While her husband would work on the house, the woman would landscape and take unique stones out of the railroad bed. She did not wear gloves while working in the soil. She would often keep a cup of drinking water with her while she worked in the yard and developed a sore and accompanying rash at the side of her mouth that persisted several years.

Beginning in 1993, the woman conferred with her physician about certain health problems. Initially she complained of swollen lymph nodes, poor memory, and fatigue. On subsequent visits, she complained of musculoskeletal pain, which was diagnosed as fibromyalgia in 1998. Her feeling of fatigue continued through 1999, when she developed hyperkeratosis (a thickening of her skin) and skin lesions. The doctor recommended analytical testing of a hair sample. The concentration of arsenic in the hair sample was 0.12 parts per million (ppm). The reference range provided by the laboratory was less than 0.06 ppm. The resident underwent chelation therapy.

Another female resident was diagnosed with leukemia in 2000. She and her husband have owned their vacation home by Crystal Lake for 36 years and currently spend more time there than at their home downstate. Reportedly, the woman spends an "average" amount of time gardening on the property. She and her husband had relocated a portion of the railroad bed to another area in their yard prior to her leukemia diagnosis.

Because the company that owned and operated the railroad no longer exists, the Michigan Department of Transportation (MDOT) has become legal custodian of the railroad and its right-of-way. The Michigan Department of Natural Resources (MDNR) is responsible for coordinating the development of trails on state land. The MDEQ is responsible for protection of the public health and natural resources through regulatory authority and remedial action. In March 2002, MDCH wrote to the MDEQ Environmental Response Division, suggesting that MDEQ, MDOT, and MDNR work together to address the contamination issue. The four agencies began discussions April 9, 2002, with subsequent meetings June 18 and September 20, 2002.

DISCUSSION

The environmental sampling results discussed in this consultation were taken from the available investigations of the property, and are not adjusted for limitations or bias in the sampling programs.

The soil sample results were compared to the MDEQ Residential Direct Contact Criterion (DCC) for the corresponding chemical. The DCC represents a level in soil that protects people from unacceptable exposure to contaminants through incidental (unintentional) ingestion of and dermal (skin) contact with soil. The Residential DCC is protective of both adults and children who might live on an affected property.

The groundwater sample results were compared to the MDEQ Residential Drinking Water Criterion (DWC) or, if more protective of human health, to the Generic Groundwater Surface Water Interface Criterion (GSI) for the corresponding chemical. The DWC identifies a drinking water (groundwater or surface-water derived) concentration that is safe for long-term, daily consumption. The GSI identifies a groundwater concentration that is protective of a surface water into which that groundwater might flow. The GSI is based on the most protective concentration for either aquatic life, terrestrial wildlife, or human health (which considers incidental ingestion of contaminated surface water and consumption of contaminated fish).

Environmental Contamination

In May 1999, MDEQ collected six soil samples at a depth of 0 to 6 inches from the railroad bed and analyzed them for the "Michigan 10" metals(1) and for polynuclear aromatic compounds (PNAs)(2). The samples were taken from the middle of the railroad bed. Results indicated that, except for arsenic, all the metals were below their corresponding MDEQ Residential DCC. The range of arsenic concentrations was 8.4 to 72 ppm. The Residential DCC for arsenic is 7.6 ppm (MDEQ 2002a). All of the soil samples had elevated detection limits reported for the PNA analyses, suggesting that there may have been matrix interferences and limiting the reliability of the data. Matrix interferences can occur when soil is contaminated with petroleum products, such as creosote from railroad ties. The PNA detection limits reported were equal to or exceeded the Residential DCC for benzo(a)pyrene (2 ppm) and dibenz(a,h)anthracene (2 ppm) (MDEQ 2002b,c). Therefore, it could not be concluded that these two PNAs were indeed present in the soil or at the concentrations reported.

As follow-up to the initial analyses, MDEQ returned to the sampling locations in June 1999 and collected additional soil samples as well as groundwater samples from two monitoring wells. Two to four additional soil samples were taken from each of four original sampling locations, at a lateral distance ranging from 5 to 20 feet from the original spot, extending along a transect to the outside of the railroad bed into the residential yards. A total of 13 additional soil samples were collected. Four samples contained arsenic above the Residential DCC, ranging from 9 to 70 ppm. Two samples had elevated detection limits reported for the PNA analyses, with the limits equal to or exceeding the Residential DCC for benzo(a)pyrene and dibenzo(a,h)anthracene, making the actual presence or concentrations of these compounds questionable. (These two samples corresponded to the two highest arsenic concentration locations.) The remainder of the samples had acceptable detection limits for PNAs, and there were no exceedances of the Residential DCC for those compounds. The concentrations of metals and PNAs in the groundwater were also within acceptable limits.

In August 2001, MDEQ collected six soil samples from the residential property belonging to the woman with the skin condition described above. (Her property had not been tested previously.) All of the samples contained elevated levels of arsenic, ranging from 9.8 to 48 ppm. Two samples had elevated detection limits reported for the PNA analyses, with the limits equal to or exceeding the Residential DCC for dibenzo(a,h)anthracene, again making the presence or concentration of this compound questionable. However, the benzo(a)pyrene concentrations in these two samples (3.7 and 8.8 ppm) were above the detection limit, indicating that the presence of this compound was likely, though the reported concentration remained questionable. The remainder of the samples had acceptable detection limits for PNAs, and there were no exceedances of the Residential DCC for those compounds.

In May 2002, in order to determine the depth of the contamination in the railroad bed and if portions of the bed soil moved by the residents contained arsenic, the MDEQ analyzed 11 additional soil samples. Some samples were paired, with one sample containing the top few inches of dirt and the other, from the same location, taken at a depth of 1.5 to 2 feet. Elevated levels of arsenic were detected only in samples taken from the top layer of soil. The samples taken at a depth of 1 foot or greater did not contain levels of arsenic in excess of the Residential DCC. Detections of PNAs also occurred only in samples taken from the top layer of soil. Two PNA samples had levels of benzo(a)pyrene above the Residential DCC for that compound, at 3.5 and 4.6 ppm. (Four samples had detections.) Four samples had detections of dibenzo(a,h)anthracene but none exceeded the Residential DCC for that compound. The detection limits for these samples were within acceptable values, indicating that the results were reliable.

In June 2002, in order to assure that there was no contamination of the water supply to the homes adjacent to the Crystal Lake Bike Trail, MDEQ sampled the private wells at 10 houses along the trail. There were no exceedances of the Residential DWC for metals or the PNAs.

It should be noted that the soil samples were taken as discrete, not composite, samples. It is possible that the sampling protocol was biased in order to determine the extent of contamination. If so, the analytical results do not represent the average of each yard, which is likely to be lower. The Agency for Toxic Substances and Disease Registry (ATSDR) and MDCH prefer to determine the likelihood of health effects based on an average concentration (which is usually more representative of exposures), but ATSDR and MDCH we consider all information available when assessing a site.

Human Exposure Pathways

To determine whether nearby residents are, have been, or are likely to be exposed to contaminants associated with a property, ATSDR and MDCH evaluate the environmental and human components that could lead to human exposure. An exposure pathway contains five elements: (1) a source of contamination, (2) contaminant transport through an environmental medium, (3) a point of exposure, (4) a route of human exposure, and (5) an exposed population. An exposure pathway is considered complete if there is evidence that all five of these elements are, have been, or will be present at the property. Alternatively, an exposure pathway is considered complete if there is a high probability of exposure. It is considered either a potential or an incomplete pathway if there is no evidence that at least one of the elements above are, have been, or will be present at the property, or that there is a lower probability of exposure.

The source of arsenic contamination along the Crystal Lake Bike Trail stems from the historic use of arsenical pesticides by the railroad company for weed control and may also come from the ballast in (rocks used in the construction of) the railroad bed. The source of benzo(a)pyrene contamination along the trail stems from the use of creosote-treated lumber for railroad ties and may also be a result of incomplete combustion of coal or diesel fuel used by the locomotive formerly operated by the railroad company. The environmental medium transporting these chemicals is the soil in the railroad bed. Exposure occurs primarily where people are in contact with soil, such as when landscaping or walking on the railroad bed, but may also occur if people consume food or handle articles that have contaminated dust on them. The chemicals can enter the body via incidental (unintentional) ingestion, dermal absorption, or inhalation of dusts. Residents and vacationers represent the potentially exposed population.

Toxicological Evaluation

Arsenic

Arsenic is a naturally occurring element widely distributed in the earth's crust. Some nutritional studies indicate that arsenic may be a nutrient essential for good health. Inorganic arsenic compounds are used mainly to preserve wood. Organic arsenic compounds are used as pesticides, primarily on cotton plants (ATSDR 2000).

The ATSDR uses Comparison Values for chemicals of interest to eliminate those chemicals in contaminated media that are not expected to cause adverse effects and retain those that need further evaluation. Comparison Values are estimated concentrations of chemicals in specific media and are based on standardized ingestion or inhalation rates and body weights. The concentrations are calculated from scientific literature available on exposure and health effects observed in laboratory animals and/or humans. The ATSDR Environmental Media Evaluation Guide (EMEG) for arsenic in soil is 20 ppm for a child and 200 ppm for an adult (ATSDR 2002). If the potentially exposed population at this site were only adults, then further evaluation of the exposure scenario would not be necessary. However, because children are likely to be present at this site, and because an adult resident was diagnosed with arsenic toxicity, further evaluation is warranted.

Perhaps the single most common and characteristic sign of oral exposure to inorganic arsenic is the appearance of skin ailments which include: hyperkeratinization (thickening) of the skin, especially on the palms and soles; formation of multiple hyperkeratinized corns or warts; and hyperpigmentation (darkening, usually a speckled pattern) of the skin with some hypopigmentation (loss of pigmentation). These effects are usually the earliest observable signs of chronic (long-term) exposure to arsenic (ATSDR 2000). These ailments are not generally found due to exposure to arsenic levels such as those found at this site.

Direct dermal contact with arsenic might cause local irritation and contact dermatitis. The effects can be mild but might progress to papules (inflamed pimples) and vesicles (blisters or cysts) in extreme cases. Still, these symptoms have only been observed in workplace environments when workers were exposed to high levels of arsenic in dust. They have not been documented in people exposed dermally to arsenic in soil (ATSDR 2000).

Other symptoms of chronic arsenic toxicity can include sensory effects, such as particularly painful dysesthesia (an unpleasant, abnormal sensation), which occur earlier in the progression of symptoms. A reversible bone marrow depression can occur. Anemia is common in chronic arsenic toxicity as well (ATSDR 1990). Again, these ailments are not generally found due to exposure to arsenic levels such as those found at this site.

Inorganic arsenic has been classified as a human carcinogen (EPA 1998). The ATSDR Cancer Risk Evaluation Guide (CREG) is a Comparison Value used to evaluate concentrations of carcinogens in specific media that are unlikely to result in an increase of cancer rates in an exposed population (no more than one additional cancer in one million exposed adults). The value is only applicable to adult exposure, not to childhood exposure. The CREG for arsenic in soil is 0.5 ppm (ATSDR 2002). The soil samples taken from the Crystal Lake Bike Trail site exceed this value. Therefore, further evaluation is warranted.

As stated in the Environmental Contamination section, the soil samples were taken as discrete, not composite, samples. It is likely that the sample results represent the maximum concentrations of arsenic to be found in each residential yard and that the remaining soil on each property contains less, if any, arsenic. If the rail bed were to remain undisturbed, persons would have only intermittent exposure to that soil and health effects would not be expected. However, several homeowners reportedly have relocated portions of the rail bed in their respective yards (2002, P. Ollila, Michigan Department of Transportation, personal communication) or, like the resident with the skin ailment, have gardened or landscaped in or near the bed. Therefore, it is not known to what degree persons might have been or might be exposed to arsenic-contaminated soil. Nonetheless, the likelihood that the high concentrations of arsenic became diluted upon relocation of the soil and the brief exposure period during these activities suggest that adverse health effects would not be expected.

Several studies have shown that ingestion of arsenic in drinking water can increase the risk of lung, bladder, liver, kidney, skin, or prostate cancer (ATSDR 2000). That said, however, unsafe levels of arsenic have not been found in the drinking water in the Crystal Lake Bike Trail area. Breathing dust that contains high levels of arsenic, such as would be found in an industrial setting, might increase the risk of lung cancer (ATSDR 2000). But any arsenic-containing dust generated in the Crystal Lake Bike Trail area would likely have lower concentrations and thus would not likely present a risk.

PNAs

PNAs are part of a group of chemicals known as polycyclic aromatic hydrocarbons (PAHs). PAHs are a group of over 100 different chemicals formed during the incomplete burning of coal, oil and gas, garbage, or other organic substances like tobacco or charbroiled meat. Some PAHs are manufactured. PAHs are usually found as mixtures in coal tar, crude oil, creosote, and roofing tar, but a few are used in medicines or to make dyes, plastics, and pesticides (ATSDR 1995).

There is no ATSDR EMEG for benzo(a)pyrene, nor is there a U.S. EPA Reference Dose (RfD). An RfD is an estimate of daily oral exposure to a substance that is likely to be without a discernable risk of adverse health effects during a lifetime of exposure. Because these values are not available, further evaluation of benzo(a)pyrene at this site is warranted.

Animal studies have shown that PAHs can cause harmful effects on the skin, body fluids, and ability to fight disease after both short- and long-term exposure. These effects have not been seen in people, however (ATSDR 1995). Chronic exposure to benzo(a)pyrene in humans has resulted in dermatitis and photosensitization in sunlight (EPA 2002).

Some PAHs have caused cancer, including leukemia, in laboratory animals (ATSDR 1995). Some people who have breathed or touched mixtures of PAHs and other chemicals for long periods of time have developed cancer. Benzo(a)pyrene has been classified as a probable human carcinogen (EPA 1994). The ATSDR CREG for benzo(a)pyrene is 0.1 ppm (ATSDR 2002). The soil samples taken from the Crystal Lake Bike Trail site exceed this value. Therefore, further evaluation is warranted.

As stated in the Environmental Contamination section, the soil samples were taken as discrete, not composite, samples. It is likely that the sample results represent the maximum concentrations of benzo(a)pyrene to be found in each residential yard and that the remaining soil on each property contains less, if any, of the compound. If the rail bed were to remain undisturbed, persons would have only intermittent exposure to that soil and health effects would not be expected. However, as discussed earlier, several homeowners have relocated portions of the rail bed in their yards (2002, P. Ollila, Michigan Department of Transportation, personal communication) or, like the resident with the skin ailment, have gardened or landscaped in or near the bed. Therefore, it is not known to what degree persons might have been or might be exposed to benzo(a)pyrene-contaminated soil. Nonetheless, the likelihood that the concentrations of benzo(a)pyrene became diluted upon relocation of the soil and the brief exposure period during these activities suggest that adverse health effects would not be expected.

ATSDR Child Health Initiative

Children may be at greater risk than adults from exposure to hazardous substances at sites of environmental contamination. Children engage in activities such as playing outdoors and exhibit hand-to-mouth behaviors that could increase their intake of hazardous substances. They are shorter than most adults, and therefore breathe dust, soil, and vapors closer to the ground. Their lower body weight and higher intake rate results in a greater dose of hazardous substance per unit of body weight. The developing body systems of children can sustain permanent damage if toxic exposures are high enough during critical growth stages. Even before birth, children are forming the body organs they need to last a lifetime. Injury during key periods of growth and development could lead to malformation of organs (teratogenesis), disruption of function, and premature death. Exposure of the mother could lead to exposure of the fetus, via the placenta, or affect the fetus because of injury or illness sustained by the mother (ATSDR 1998). The clear implication for environmental health is that children can experience substantially greater exposures than adults to toxicants that are present in soil, water, or air.

Children are highly likely to be exposed to the soil in the railroad bed. Some of the sections of the bed are next to or even under the beach, where children would play in the sand. Similar to the first resident described above, children might also dig for stones in the bed. Children who display "pica" behavior, the condition of intentionally eating non-food substances such as soil, may be exposed to a higher overall dose of arsenic. However, exposure would occur only during the warmer months and not year-round.

COMMUNITY HEALTH CONCERNS

First Resident

The symptoms experienced by the first resident are consistent with what could occur if a sensitized individual were exposed to excess arsenic. Although the magnitude of the difference between the DCC and maximum soil concentration of arsenic on the woman's property may not be considered great, it is possible that the woman is more susceptible to the toxic effects of arsenic than is considered the average. However, the levels found at this site are lower than those that would prompt concern for adverse affects in the general population. The potential for exposure from other sources/activities should be explored further. Testing of the well serving the resident's other home in Oakland County showed no elevations of arsenic in the water, eliminating a potential source of exposure. Oakland County is in an area of Michigan where the underlying bedrock can be a source of arsenic in groundwater.

Biological indicators of exposure to arsenic can be measured in blood, hair, nails, and urine. Blood levels of arsenic, however, are not reliable indicators of chronic exposure because the concentrations clear within a few hours. The metal accumulates in hair and nails, providing an indication of past exposure. However, exogenous arsenic (arsenic that is not within the body) may adhere to the cuticle of the hair or may be present in hair-care products and be absorbed into the hair follicle, or may be present in dirt on the nails, skewing analytical results. Urinary arsenic levels are accepted as the most reliable indicator of recent arsenic exposure via ingestion or inhalation (ATSDR 2000). However, because the symptoms and subsequent diagnosis occurred more than five years ago, a urine test now will not be useful in trying to corroborate with her physician's diagnosis then.

It is possible that the benzo(a)pyrene in the railroad bed soil could have contributed to the dermatitis experienced by the woman. As well, it could have caused the woman to become photosensitive.

Second Resident

Although the second resident and her husband have used their property for nearly 40 years and currently spend a majority of their time there, the data do not suggest that the contamination of the soil is at a sufficient level to have caused her illness.

Other Concerns

A citizen contacted MDCH by e-mail, expressing her concerns that the wild-growing black raspberries she and her family pick along the railroad bed could be contaminated. She was looking for a laboratory that would analyze the berries. The MDCH toxicologist conferred with a colleague in the Michigan Department of Agriculture regarding laboratories that analyze for pesticide residues and relayed that information to the citizen. In addition, the toxicologist discussed in the reply that there should be more concern over what might be on the berries rather than in them, recommending that the citizen wash the berries and her hands prior to eating in order to remove any contaminated dust.

CONCLUSIONS

Because the magnitude of difference between the state screening level and concentrations of arsenic found in soil samples is not great, and because exposure is likely to be intermittent, adverse health effects are not expected to occur as a result of exposure to soil at this site. The concentrations of arsenic and benzo(a)pyrene in the Crystal Lake Bike Trail railroad bed that passes through residential properties pose no apparent public health hazard. One citizen exposed to the railroad bed soil did display symptoms that can result from chronic arsenic exposure and was diagnosed by her doctor as having arsenic toxicity. The ATSDR and MDCH do not negate the diagnosis of an individual's personal physician, who would have a more comprehensive understanding of the medical history of that individual.

RECOMMENDATIONS

In order to minimize potential further exposures, and because soil sampling results were greater than the state clean-up criteria for arsenic and benzo(a)pyrene, the contamination of the soil and ballast of the railroad bed should be addressed, with remediation being an option. The residential community should be kept apprised of any remedial process and be educated about ways to reduce or eliminate exposure to the chemicals in the railroad bed.

Public Health Action Plan

  • The MDEQ, MDOT, and MDNR will work cooperatively, with MDEQ having regulatory leadership, to address the contamination of the soil and ballast along residential portions of the Crystal Lake Bike Trail. The MDCH will attend any interagency meetings in an advisory capacity. (This action is currently in progress.)


  • The MDEQ will compose an information bulletin regarding the background of the investigation and any remedial process, for distribution to the citizens in the affected area. The MDCH will supplement the bulletin with public health and safety information regarding the contamination. (This bulletin was distributed to the residents at a July 2002 meeting.) Additional information will be provided to the citizens as necessary.

If any citizen has additional information or health concerns regarding this health consultation, please contact the Michigan Department of Community Health, Environmental and Occupational Epidemiology Division, at 1-800-648-6942.

PREPARERS OF REPORT

Michigan Department of Community Health

Christina Bush, Toxicologist

Robin Freer, Resource Specialist

ATSDR Regional Representative

Mark Johnson
Regional Services, Region V
Office of the Assistant Administrator

ATSDR Technical Project Officer

Alan Yarbrough
Division of Health Assessment and Consultation
Superfund Site Assessment Branch

REFERENCES

Agency for Toxic Substances and Disease Registry (ATSDR). 1995. Toxicological Profile for Polycyclic Aromatic Hydrocarbons.

ATSDR. 2000. Toxicological Profile for Arsenic.

ATSDR. 2002. Soil Comparison Values.

Michigan Department of Environmental Quality (MDEQ). 2002a. Generic Residential and Commercial I Direct Contact Criteria. Arsenic.

MDEQ. 2002b. Generic Residential and Commercial I Direct Contact Criteria. Benzo(a)pyrene.

MDEQ. 2002c. Generic Residential and Commercial I Direct Contact Criteria. Dibenzo(a,h)anthracene.

U.S. Environmental Protection Agency (EPA). 1994. Integrated Risk Information System. Benzo(a)pyrene.

U.S. EPA. 1998. Integrated Risk Information System. Inorganic arsenic.

U.S. EPA. 2002. Technology Transfer Network. Unified Air Toxics Website. Polycyclic Organic Matter.

Map of Affected Area - Southeast Shore of Crystal Lake
Figure 1. Map of Affected Area - Southeast Shore of Crystal Lake

CERTIFICATION

This Crystal Lake Bike Trail Health Consultation was prepared by the Michigan Department of Community Health under a cooperative agreement with the Agency for Toxic Substances and Disease Registry (ATSDR). It is in accordance with approved methodology and procedures existing at the time the health consultation was begun.

Alan W. Yarbrough
Technical Project Officer, SPS, SSAB, DHAC, ATSDR

The Division of Health Assessment and Consultation, ATSDR, has reviewed this public health consultation and concurs with the findings.

Roberta Erlwein
Chief, State Programs Section, SSAB, DHAC,ATSDR

1 Michigan 10 metals are arsenic, barium, cadmium, chromium, copper, lead, mercury, selenium, silver, and zinc.
2 The PNAs tested for were acenaphthene, acenaphthylene, anthracene, benzo(a)anthracene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(g,h,i)perylene, benzo(a)pyrene, chrysene, dibenzo(a,h)anthracene, fluoranthene, fluorene, indeno(1,2,3-cd)pyrene, 2-methylnaphthalene, naphthalene, phenanthrene, and pyrene.


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